The differential inhibition of hemopoietic growth factor activity by cytotoxins and interferon-gamma

The effects of recombinant human tumor necrosis factor (TNF), lymphotoxin (LT), and interferon-gamma (IFN-gamma) on the growth of human hemopoietic progenitor cells in clonal culture have been examined. Colony growth was induced by using granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). A suppressive effect of TNF, LT, and IFN-gamma on the development of granulocyte, macrophage, and mixed granulocyte/macrophage colonies was shown. Suppression of colonies formed after stimulation with G-CSF was greater than that observed after stimulation with GM-CSF. In the presence of a monoclonal antibody to TNF, or polyclonal antibodies to either LT or IFN-gamma, the inhibitory effect of the molecule to which the antibody was directed was abrogated. These findings suggest that progenitor cells responsive to G-CSF or GM-CSF have different sensitivities to the effects of TNF, LT, and IFN-gamma. Defining the interactions of growth factors and inhibitors should increase understanding of mechanisms underlying diseases associated with suppression of normal hemopoiesis, and in predicting the effects in vivo of these bioregulatory molecules in clinical medicine.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

Effect of cytokines on Japanese encephalitis virus production by human monocytes

Japanese encephalitis (JE) virus was shown to grow in in vitro cultures of human monocytes. Interferon (IFN)-alpha and IFN-gamma inhibited JE virus production by the infected monocytes in the absence of anti-JE virus antibody, but interleukin (IL)-1 alpha, IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and tumor necrosis factor (TNF)-alpha did not show a significant inhibition. Antibody against JE virus increased the JE virus production by the infected monocytes probably by enhanced uptake of virus-antibody complexes via Fc receptors. IFN-gamma and GM-CSF increased JE virus production by monocytes in the presence of anti-JE virus antibody, whereas IFN-alpha inhibited JE virus production even in the presence of the antibody. The other 5 cytokines (IL-1 alpha, IL-2, IL-3, G-CSF, and TNF-alpha) did not show a significant effect on JE virus production by monocytes in the presence or absence of the antibody.     

Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.     

Recombinant murine granulocyte-macrophage (GM) colony-stimulating factor supports formation of GM and multipotential blast cell colonies in culture: comparison with the effects of interleukin-3

We studied the effects of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on murine hemopoiesis in methylcellulose culture. The GM-CSF was purified from cultures of Saccharomyces cerevisiae transfected with a cloned murine GM-CSF cDNA. In cultures of spleen cells from normal mice, only granulocyte-macrophage (GM) colonies were supported by GM-CSF. Blast cell colonies were the predominant type in cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice. Dose-response studies revealed that maximal GM and blast cell colony formation is achieved with 100 U/ml GM-CSF. Blast cell colonies revealed variable but high replating efficiencies, and the secondary colonies included multilineage colonies. Serial replating of washed blast cell colonies in cultures with GM-CSF provided evidence for the direct effects of GM-CSF on the proliferation of multipotential blast cells. A combination of GM-CSF and interleukin-3 (IL-3) did not increase the number of blast cell colonies over the level supported by IL-3. This observation indicates that the progenitors for blast cell colonies that responded to GM-CSF are a subpopulation of multipotential progenitors that are supported by IL-3. Cytological studies of colonies derived from GM-CSF and/or IL-3 suggest that the eosinophilopoietic ability of murine GM-CSF is less than that of IL-3.     

Severe Leukopenia and Dysregulated Erythropoiesis in SCID Mice Persistently Infected with the Parvovirus Minute Virus of Mice

Parvovirus minute virus of mice strain i (MVMi) infects committed granulocyte-macrophage CFU and erythroid burst-forming unit (CFU-GM and BFU-E, respectively) and pluripotent (CFU-S) mouse hematopoietic progenitors in vitro. To study the effects of MVMi infection on mouse hemopoiesis in the absence of a specific immune response, adult SCID mice were inoculated by the natural intranasal route of infection and monitored for hematopoietic and viral multiplication parameters. Infected animals developed a very severe viral-dose-dependent leukopenia by 30 days postinfection (d.p.i.) that led to death within 100 days, even though the number of circulating platelets and erythrocytes remained unaltered throughout the disease. In the bone marrow of every lethally inoculated mouse, a deep suppression of CFU-GM and BFU-E clonogenic progenitors occurring during the 20- to 35-d.p.i. interval corresponded with the maximal MVMi production, as determined by the accumulation of virus DNA replicative intermediates and the yield of infectious virus. Viral productive infection was limited to a small subset of primitive cells expressing the major replicative viral antigen (NS-1 protein), the numbers of which declined with the disease. However, the infection induced a sharp and lasting unbalance of the marrow hemopoiesis, denoted by a marked depletion of granulomacrophagic cells (GR-1⁺ and MAC-1⁺) concomitant with a twofold absolute increase in erythroid cells (TER-119⁺). A stimulated definitive erythropoiesis in the infected mice was further evidenced by a 12-fold increase per femur of recognizable proerythroblasts, a quantitative apoptosis confined to uninfected TER-119⁺ cells, as well as by a 4-fold elevation in the number of circulating reticulocytes. Therefore, MVMi targets and suppresses primitive hemopoietic progenitors leading to a very severe leukopenia, but compensatory mechanisms are mounted specifically by the erythroid lineage that maintain an effective erythropoiesis. The results show that infection of SCID mice with the parvovirus MVMi causes a novel dysregulation of murine hemopoiesis in vivo.     

Granulocyte-macrophage colony stimulating factor induces both HLA-DR expression and cytokine production by human monocytes

The effect of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) on the expression of HLA-DR, and the production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) by human peripheral blood monocyte-enriched populations was investigated. GM-CSF was shown to induce both the expression of HLA-DR and the cytokines IL-1 and TNF alpha in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma), which induced major histocompatibility complex (MHC) class II expression, did not induce IL-1 or TNF alpha production. However, IFN-gamma enhanced the cell surface expression of HLA-DR and the production of IL-1 and TNF alpha on monocyte-enriched cells stimulated by GM-CSF. By itself, GM-CSF did not induce surface class II expression on the human monocytic tumour cell line THP-1, whereas it synergized with IFN-gamma to induce surface expression. These cells responded to GM-CSF by producing IL-1 and TNF alpha; Northern blotting showed that mRNA levels of IL-1 and TNF alpha were transiently induced, similar to other cytokines. Our results indicate that GM-CSF is a major macrophage activating factor that is capable of inducing both the expression of HLA-DR and the cytokines involved in T-cell activation by macrophages; therefore, GM-CSF may be of importance in potentiating antigen presenting function.     

IL-7 induces myelopoiesis and erythropoiesis

IL-7 administration to mice was previously reported to increase the mobilization of progenitor cells from marrow to peripheral sites. We now report that IL-7 increases the number of mature myeloid and monocytic cells in spleen and peripheral blood. This effect required T cells, and we show that IL-7 treatment in vivo induced GM-CSF and IL-3 production by T cells with memory phenotype. However, additional myelopoietic cytokines were shown to be involved because mice deficient in both GM-CSF and IL-3 also responded to IL-7 with increased myelopoiesis. Candidate cytokines included IFN-gamma and Flt3 ligand, which were also produced in response to IL-7. Because IFN-gamma-deficient mice also increased myelopoiesis, it was suggested that IL-7 induced production of redundant myelopoietic cytokines. In support of this hypothesis, we found that the supernatant from IL-7-treated, purified T cells contained myelopoietic activity that required a combination of Abs against GM-CSF, IL-3, and anti-Flt3 ligand to achieve maximum neutralization. IL-7 administration increased the number of splenic erythroid cells in either normal, Rag1 or GM-CSF-IL-3-deficient mice, suggesting that IL-7 might directly act on erythroid progenitors. In support of this theory, we detected a percentage of TER-119(+) erythroid cells that expressed the IL-7Ralpha-chain and common gamma-chain. Bone marrow cells expressing IL-7R and B220 generated erythroid colonies in vitro in response to IL-7, erythropoietin, and stem cell factor. This study demonstrates that IL-7 can promote nonlymphoid hemopoiesis and production of cytokines active in the host defense system in vivo, supporting its possible clinical utility.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum-replaced cultures

Erythroid progenitors from normal human marrow were purified by a two-step immune panning method permitting both the enrichment of erythroid progenitors (plating efficiency up to 10%) and the separation of CFU-E from BFU-E. The purified erythroid progenitors were grown in serum-replaced conditions; in some experiments at an average of one cell per well. Human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL3), erythroid potentiating activity (EPA), and human erythropoietin (Epo) either recombinant or homogenous native were tested for their effect on CFU-E growth. Epo was an absolute requirement for CFU-E growth and was sufficient to obtain colony formation at the unicellular level whereas GM-CSF and IL3 did not further increase the plating efficiency. EPA potentiated the effect of Epo on this progenitor only in experiments performed at unicellular level. Human recombinant GM-CSF, IL3, Interleukin 1 alpha (IL1 alpha), and Epo were subsequently tested for their ability to promote BFU-E growth. GM-CSF and IL3 supported the growth of erythroid bursts in the presence of Epo, even at the unicellular level. However, IL3 promoted a higher number of bursts than GM-CSF under all conditions tested. These two growth factors have no or very small additive effects when tested in combination. IL1 alpha added to Epo alone had no effect on the growth of BFU-E whereas it potentiated the combined action of IL3 and GM-CSF on the primitive BFU-E. In conclusion, this study confirms at the unicellular level and under serum-free conditions that erythroid progenitors are regulated by multipotential growth factors in early phases of erythropoiesis and become sensitive only to Epo in later phases of differentiation.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on single CD34-positive hemopoietic progenitors from human bone marrow

To determine the extent accessory cells mediate the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on human hemopoietic progenitors in vitro, we added this hemopoietin to liquid cultures of single CD34-positive marrow cells. These were selected on a fluorescence-activated cell sorter using the HPCA-1 (My10) antibody. Myeloid, erythroid and a few mixed clones developed in 13% of wells in the apparent absence of accessory cells at the beginning of culture. Although accessory cells were generated quickly from the myeloid progenitors and could have mediated the action of rhGM-CSF, this was not the case in the majority of the erythroid clones in which no other cell types were recorded. We conclude that rhGM-CSF can act directly on a subset of erythroid progenitors and probably induces a substantial number of myeloid clones directly.     

Regulatory effect of cytokines on eosinophil degranulation

We tested the effects of different cytokines on IgA- and IgG-induced eosinophil degranulation in vitro to determine the potential interaction between eosinophils and mononuclear cells. Purified normodense eosinophils were incubated with cytokines (including rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, IFN-gamma, granulocyte-macrophage CSF stimulating factor (GM-CSF), and TNF) for 1 to 3 h after which Ig-coupled Sepharose 4B beads were added as targets and the mixtures were incubated with the eosinophils at 37 degrees C for 4 h. The Ig used were secretory IgA (sIgA), serum IgA and IgG, and myeloma IgA and IgG. The release of eosinophil-derived neurotoxin (EDN) was measured by RIA as an index of degranulation. rIL-5 was the most potent enhancer of Ig-induced degranulation and increased EDN release by 48% for sIgA and 136% for IgG. The effect of rIL-5 appeared as quickly as 15 min after incubation of eosinophils, sIgA beads and IL-5. GM-CSF and rIL-3 also enhanced Ig-induced EDN release but less potently than rIL-5. GM-CSF and rIL-5 by themselves induced a small but significant release of EDN from eosinophils in the absence of Ig-coated beads; rIL-3 did not. However, IFN-gamma suppressed sIgA-induced EDN release by 23%. The other cytokines did not have any effect on eosinophil degranulation. These results suggest that cytokines which induce eosinophil differentiation and proliferation during hematopoiesis also enhance the effector function of mature eosinophils and that IFN-gamma partially down-regulates eosinophil degranulation.     

Cutting edge: activation of the p38 mitogen-activated protein kinase signaling pathway mediates cytokine-induced hemopoietic suppression in aplastic anemia

Myelosuppressive cytokines, in particular IFN-gamma and TNF-alpha, play an important role in the pathogenesis of idiopathic aplastic anemia in humans. It is unknown whether these negative regulators of hemopoiesis suppress stem cells by activating a common signaling cascade or via distinct nonoverlapping pathways. In this study, we provide evidence that a common element in signaling for IFN-gamma and TNF-alpha in human hemopoietic progenitors is the p38/MapKapK-2 signaling cascade. Our studies indicate that pharmacological inhibition of p38 reverses the suppressive effects of IFN-gamma and TNF-alpha on normal human bone marrow-derived erythroid and myeloid progenitors. Most importantly, inhibition of p38 strongly enhances hemopoietic progenitor colony formation from aplastic anemia bone marrows in vitro. Thus, p38 appears to play a critical role in the pathogenesis of aplastic anemia, suggesting that selective pharmacological inhibitors of this kinase may prove useful in the treatment of aplastic anemia and other cytokine-mediated bone marrow failure syndromes.     

IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice

Incubation of the human U937 histiocytic lymphoma cell line with granulocyte-macrophage colony stimulating factor (GM-CSF) rendered the cells responsive to induction of TNF by LPS. Treatment with IL-6 reduced TNF production in GM-CSF-primed U937 cells. The inhibitory effect was most pronounced (approximately equal to 80%) when IL-6 was added either along with GM-CSF or within the first 3 h of GM-CSF treatment. Both GM-CSF or IL-6 inhibited [3H]TdR uptake in U937 cells, and simultaneous treatment with GM-CSF and IL-6 resulted in an additive inhibitory effect on cell proliferation. However, the inhibition of TNF production could not be explained by the inhibitory effect of IL-6 on cell growth, nor was it due to a reduction in cell viability. An inhibition of TNF production by IL-6 was also demonstrated in cultured human peripheral blood monocytes. Treatment with IL-6 also resulted in a dose-dependent reduction of the 17-kDa TNF band revealed by SDS-PAGE after labeling monocytes with [35S]cysteine and immunoprecipitation with anti-TNF mAb. In addition, treatment with IL-6 resulted in a reduction of monocyte in vitro cytotoxicity for tumor target cells. Finally, in mice sensitized by the administration of Bacillus Calmette-Guérin, the injection of IL-6 significantly reduced the levels of TNF found in the serum upon challenge with LPS. Inasmuch as TNF is known to be an inducer of IL-6, the inhibitory action of IL-6 on TNF production may represent the negative arm of a regulatory circuit. The inhibitory action of IL-6 on TNF production is consistent with a predominantly antiinflammatory role of IL-6 in the intact organism.     

Lithium chloride potentiates tumor necrosis factor-induced and interleukin 1-induced cytokine and cytokine receptor expression.

The antimalignant cell activity of tumor necrosis factor (TNF) in many cell types can be enhanced by lithium chloride (LiCl). This study shows the in vitro effect of LiCl on the TNF-induced or interleukin 1 (IL-1)-induced expression of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-2, and the IL-2 receptor-alpha (IL-2R alpha). The levels of IL-6 and GM-CSF in the medium of TNF-treated L929 fibrosarcoma cells were increased by cotreatment with LiCl. In contrast, enhancement of IL-6 production by dibutyryl cyclic AMP or cycloheximide was not affected by LiCl. The production of IL-6 and GM-CSF was not correlated with sensitivity to TNF-mediated cell killing. IL-1 by itself had no measurable effects on L929 cells. However, LiCl potentiated the IL-1-induced synthesis of IL-6, GM-CSF, IL-3, and IL-2 in PC60 murine T-cell hybridoma cells. TNF alone induced only GM-CSF production in these cells, but in the presence of LiCl, increased amounts of GM-CSF as well as small amounts of IL-2 and IL-6 could be detected. It is also shown that in these PC60 cells the expression of the IL-2R alpha was induced by TNF + LiCl treatment but not by TNF alone. IL-2R alpha expression was likewise considerably enhanced by IL-1 + LiCl treatment, as compared with treatment with IL-1 alone. The effects of LiCl on the TNF-induced and the IL-1-induced gene expression seem to be independent of the protein kinase A and C pathways. These results show that LiCl can modulate both TNF-mediated cytotoxicity and TNF-induced and IL-1-induced cytokine expression, suggesting that Li+ acts early in the TNF-signaling pathway, but at a step shared with the IL-1-signaling pathway.